Characteristics of Anaplastic Oligodendrogliomas Short-Term Survivors: A POLA Network Study

Anaplastic oligodendrogliomas (AO) are rare brain tumors, accounting for approximately 5% of adult gliomas and 0.5% of all primary tumors affecting the central nervous system. This article reports results of a study conducted within the frame of the French POLA network on the characteristics of survivors of AO, comparing populations with a disease-specific survival of less than 5 years with populations with a survival 5 years or more.


Introduction
Anaplastic oligodendrogliomas (AO) are rare brain tumors, accounting for approximately 5% of adult gliomas and 0.5% of all primary tumors affecting the central nervous system. 1 They are defined by the 2016 World Health Organization (WHO) classification 2 as IDH-mutant 1p/19q-codeleted diffuse gliomas with increased mitotic activity, microvascular proliferation, and/or necrosis. 3 Among high-grade gliomas, AO have a better prognosis than high-grade IDH-mutant astrocytomas and a much better prognosis than IDH-wild-type glioblastomas. 3 Standard treatment consists of maximal safe surgical resection followed by radiotherapy plus PCV chemotherapy (CT) regimen (procarbazine, CCNU [lomustine], and vincristine). 4,5 Anaplastic oligodendrogliomas are typically associated with a prolonged survival with a median survival estimated to approximately 15 years. 4,5 However, approximately 20% of patients have a poor outcome and survive less than 5 years. [4][5][6] Poor prognostic factors have been identified in AO, [7][8][9] yet the characteristics of AO short-term survivors (STS) remain to be described. 10 The aim of the present study was to analyze the characteristics of STS, defined as patients with a disease-specific survival <5 years and to compare with those of AO patients with a survival ≥5 years (classical survivors, CS). It was conducted within the frame of the French POLA network dedicated to anaplastic oligodendroglial tumors.

POLA Network and Patients
In 2008, the French Institut National du Cancer supported the creation of a national network named "Prise en charge des OLigodendrogliomes Anaplasiques" (POLA). This network prospectively collects samples, characteristics, and outcomes of patients diagnosed with high-grade oligodendroglial tumor in French academic centers. Among the 2189 patients included in the POLA network, patients with centrally reviewed confirmation of newly diagnosed AO were prospectively included in the present study. Formalin-fixed paraffin-embedded (FFPE) tumor tissue was available for pathological and immunohistochemical investigations for all cases. Patients provided written informed consent for clinical data collection and genetic analysis according to national and POLA network policies. We retrospectively analyzed data from all patients registered in the POLA network from 2008 to 2019. The following clinical data were collected: age at diagnosis, preoperative symptoms and Karnofsky Performance Status (KPS), surgical and postoperative treatments, tumor location and characteristics, survival status, and survival time. Extent of resection (EOR) was recorded as biopsy or resection. The initial postoperative treatment strategy was classified as radiotherapy (RT) alone, CT alone, RT + CT (sequential and/or concurrent), simple follow-up, and/or no treatment. CT regimen was defined as PCV, TMZ (temozolomide), or CCNU (lomustine). Tumor volumes could be measured for a subset of 64 patients and were calculated according to the 3 largest diameter technique using T2 or FLAIR-weighted magnetic resonance imaging. 11 Pathological Review and Immunohistochemistry All cases of supposed AO were centrally reviewed and included in the prospective POLA network if they met the pathological inclusion criteria of AO according to the WHO classification of brain tumors. 2 The presence of mitoses (with mitotic index referring to the number of mitotic figures per 10 High Power Fields), marked atypia, areas of high cellularity, microvascular proliferation, and necrosis were assessed. In addition, automated immunohistochemistry was performed on 4-µm-thick FFPE sections with avidinbiotin-peroxydase complex on Benchmark XT (Ventana Medical System Inc., Tucson, AZ, USA) using the Ventana Kit including DAB reagent to search for expression of IDH1 R132H (clone H09; 1:75; Diavona), p53 (clone DO.7; 1:200; Dako), ATRX (polyclonal; Sigma), Ki67/MIB1 (clone Mib1;:100; Dako), EGFR (clone EGFR.25; 1:100; BNovocasta), and inactivating mutations in the transcriptional repression factor Capicua (CIC). EGFR positive expression was assessed using the Hirsch score as previously described. 12 P53-positive expression was considered with a cutoff at 10%.

DNA Extraction, Single Nucleotide Polymorphism Array, and Comparative Genomic Hybridization Array Procedures
Following the manufacturer's recommendations, tumor DNA was extracted from frozen tissue, or FFPE samples using the iPrep ChargeSwith Forensic Kit. Qualification and quantification of tumor DNA were fulfilled using a NanoVue spectrophotometer and gel electrophoresis, respectively. When necessary, the genomic profile was assessed using single nucleotide polymorphism (SNP) or CGH arrays, as described previously. 13 TERT mutation were also assessed as previously described. 14

Statistical Analysis
SNP and CGH array analysis were performed as previously described. 15 For arrays, genomic imbalances were classified as loss, gain, homozygous deletion, or amplification. For correlation between chromosomal arm imbalances and histological variables, the Fisher' exact test (for factors) or the Student's t test (for quantitative variables, when they were scored as positive or negative) were used. Continuous variables were compared using Mann-Whitney U test. Overall survival (OS) was defined as the time from surgery to tumorprogression-related death (patients who died from other causes were excluded from the retrospective analyses). Progression-free survival (PFS) was defined as the time from surgery to first progression or last follow-up in case of unprogressive tumor. In order to identify clinical, radiological, pathological, and/or genomic factors related to OS, survival curves were obtained according to the Kaplan-Meier method and compared using the log-rank test for univariate comparisons. Cox proportional hazards models were used for multivariate analyses and for estimating hazard ratios in survival regression models. Because of the large number of potiential explanatory variables, multivariate analysis only included all the variables with a P-value of <.02 in univariate analyses. All variables obtained were searched for prognostic significance. The final model was fit using a backward method of selection. All statistical tests were 2-sided, and the final threshold for statistical significance was P-value = .05. Analysis was performed by the Clinical Investigation Center (Inserm CIC 1431) of Besançon and was conducted using SAS for windows version 9.4.

Patient Selection
At the time of analysis, among the 519 AO patients included in the POLA network, 318 patients were alive and their follow-up was <5 years, 146 patients had a survival ≥ 5 years (and constituted the CS group) and 55 patients had a survival < 5 years. Among the latter patients, 44 patients (80%) died from tumor progression and 11 patients (20%) died from another cause (suicide n = 2, other cancer n = 2, post-operative cerebral hemorrhage n = 1, stroke n = 1, congestive heart failure n = 1, pulmonary embolism n = 1, aortic aneurysm rupture n = 1, sepsis n = 1, car crash n = 1), while their last evaluation indicated stable disease. We considered as AO STS the 44 patients with a disease-specific survival < 5 years. Patients who died from another cause than tumor progression were excluded from the analysis. The CONSORT flow diagram of patient selection for the study cohort is available in Supplementary Fig. 1. The median survival of STS patients was 2 years, the median survival of CS patients was not reached after a median follow-up of 7 years. The median PFS was 0.68 years for STS patients and 5 years in CS patients.

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The Oncologist, 2022, Vol. 27,No. 5 more frequent in STS than CS but the difference did not reach statistical significance.

Treatment
Treatment characteristics of STS and CS are summarized in Table 3. Short-term survivors less frequently underwent tumor resection than CS (61.9% vs 88.8%; P = .= .0001) and more frequently needed postoperative steroids (66% vs 42%; P = .= .009). Median time from surgery to postoperative treatment onset tended to be shorter in STS than in CS (47 vs 65 days, P = .08). After surgery, STS less frequently received the treatment that was considered as the standard treatment at the time of diagnosis than CS. Before 2012, they were less frequently treated with RT alone, and after 2012, less frequently treated with RT plus PCV (40.9% vs 63%, P = .= .01). Compared to CS, STS were more frequently treated with CT alone (22.7% vs 6.8%, P = .= .009) or radiotherapy plus TMZ (29.6% vs 15.7%, P = .= .05), and less frequently with RT alone (18.2% vs 49.3%, P = .= .0009).

Multivariate Analysis
The following characteristics were associated with the STS profile in multivariate analysis: the presence of cognitive dysfunction at diagnosis (odds ratio  Table 4). Based on these 3 characteristics, patients could be classified into groups of standard (16%) and high (61%) risk of < 5 year survival (Fig. 2).

Discussion
Although AO are frequently associated with prolonged survival, approximately 20% of patients die within 5 years after diagnosis. [4][5][6] The reason why some patients have a poor prognosis remains to be fully understood. Several studies have analyzed prognostic factors in AO but many of these studies included both 1p/19q codeleted and non-codeleted tumors as well as low-grade and high-grade oligodendrogliomas ( Table  5). To our knowledge, our study is the first one to analyze the characteristics of STS. The present study showed that although the poor prognosis of STS appears to largely result from more aggressive baseline characteristics and a more advanced disease at diagnosis, surgical resection may be a particularly important determinant of survival in these patients.

Baseline Characteristics of STS
Older age and poorer KPS have been identified as poor prognostic factors in AO across multiple studies and characterized STS in the present study. 7-9,17-32 Being aged > 60 years was associated with STS, which is consistent with a large retrospective study reporting an approximately 2 times higher median survival in 1p/19q codeleted AO patients aged < 60 years compared to those aged > 60 years. 34 In addition, we found that STS had a more aggressive clinical presentation than CS, with less frequent seizures, more frequent neurological and cognitive deficits, the latter feature was the only clinical feature independently associated with STS in multivariate analysis. Except in one study (which however included both low-and high-grade oligodendrogliomas and 1p/19q codeleted and non-codeleted tumors), the clinical presentation has not been related to prognosis in AO. 8 In contrast, neurological deficits and the absence of seizures are well-described poor prognostic factors in low-grade gliomas. 35,36 Most IDH-mutant glioma patients display seizures and it has been suggested that 2-hydroxyglutarate, the oncometabolite resulting from the IDH mutation, could explain their epileptogenicity. 37 The reason why some IDHmutant glioma patients do not display seizures remains to be determined but these patients could have a poorer outcome due to a longer time to diagnosis. Regarding radiological characteristics, STS also had a more aggressive presentation than CS. Tumors in STS were larger and more frequently presented with contrast-enhancement. Consistently, contrast-enhancement has been associated with more aggressive molecular features in AO. 38 Initial tumor   volume has not been reported as a prognostic factor in AO; however, this finding is consistent with the reported poor prognostic value of bilateral hemispheric involvement, which was in the present study one of the radiological feature associated with STS. 7 At the histo-molecular level, STS were characterized by a higher proliferative index and a number of mitosis > 8 was independently associated with STS in multivariate analysis. A higher proliferative index has been shown to be associated with a poorer outcome in AO 20,21,29,18,33 and in a recent study a radiological growth rate > 8 mm/year has been found as an independent factor of poorer PFS in oligodendrogliomas. 35 Higher proliferation index and reduced epileptogenicity could explain why STS presented larger and more symptomatic tumors in older patients. In addition, STS were characterized by a higher level of chromosomal instability compared to CS. In IDH-mutant astrocytomas, chromosomal instability is an established poor prognostic factor. 39 Its prognostic value in AO remains to be fully established, yet it has been associated with more frequent contrast-enhancement, larger tumor volume, and a poorer prognosis. 23,38 Chromosome 9p loss and CDKN2A deletion have been shown to be important poor prognostic factors in AO. 21 Herein, there was a trend toward more frequent 9p loss in STS compared to CS, but this trend was not statistically significant, possibly because of the small sample size. Other molecular alterations that have been associated with poorer outcome in AO include NOTCH1 and PI3KCA mutations, as well as specific gene expression profiles. 24,28 Yet these alterations were not assessed in the present study. Future comprehensive molecular analyses will be important to determine whether STS are characterized by specific alterations that could facilitate their identification and constitute therapeutic targets.

Treatment Characteristics of STS
Although we observed important differences regarding the treatment of STS and CS, the only treatment-related characteristic independently associated with STS in multivariate analysis was the absence of surgical resection. Surgical resection has been associated with better prognosis in several studies 7,9,17,22,23,28,32 and the present study suggests that it may be particularly important in AO patients presenting aggressive baseline characteristics. The possibility of a surgical resection should therefore be reconsidered in AO patients who have only undergone a biopsy, possibly because a diagnosis of AO was not suspected pre-operatively. Indeed, approximately 20% of AO patients have a "glioblastoma-like" presentation that may lead some teams to perform a biopsy rather than a surgical resection, especially in older patients with cognitive dysfunction. 36 After surgery, we observed that compared to CS, STS were more frequently treated with CT alone or with radiotherapy plus temozolomide. Older age and larger tumor volume could explain why STS were more frequently treated with CT alone than CS, while one can hypothesize that a more aggressive "glioblastoma-like" presentation could explain partly why STS were more frequently treated with radiotherapy plus temozolomide. Although the optimal treatment of patients at risk for poor outcome remains to be determined, CT alone, especially with temozolomide may not be the optimal treatment AO. 31,40 Whether these patients benefit from the addition of CT to radiotherapy is also unclear. Indeed, in both the RTOG and EORTC trials, survival curves of patients treated with RT plus PCV or RT only started to diverge after 5 years, as if the addition of PCV to RT had no clear impact on the outcome of AO patients at risk for poor survival. 4,5 Analysis of STS characteristics in ongoing clinical trials dedicated to AO (NCT00887146, NCT02444000) will be important to determine the impact of post-operative treatment in these patients.

Identification of STS
Identification of patients at risk for poor survival is crucial to test more effective treatment strategies in this population. Herein, combining 3 characteristics independently associated with STS (cognitive dysfunction, mitosis count, and type of surgery) enabled to distinguish 2 groups of patients with different risk of short-term survival. However, this finding needs to be validated in an independent series and future studies should try to determine baseline features that are easier to assess for the identification of STS and explore classifications. Indeed, the identification of cognitive dysfunction may depend on testing method, mitosis count may lack reproducibility, and the type of surgery performed may depend on neurosurgeons' experience.

Study Limits
Our study is limited by the absence of volumetric analysis for all patients, the absence of in-depth molecular analyses, and the heterogeneity of post-operative treatments. Because of its retrospective design, it is also difficult to determine to what extent differences regarding treatment resulted from differences in baseline characteristics and to what extent they influenced the outcome. Despite these limits, our study provides the first description of STS characteristics, highlights features that could help identifying these patients, and suggests that surgical resection may be particularly important in this population. However, these findings require validation in independent series. In addition, although cancer-specific survival may be difficult to assess in retrospective studies, our study strongly suggests that future studies on this population should carefully analyze the cause of death in poor prognosis AO patients, since here approximately 20% of the patients  who died < 5 years after diagnosis very likely died from an AO-unrelated cause. In a large series from the Surveillance, Epidemiology, and End Result (SEER) database, the rate of non-cancer death was 11.7% in adult oligodendrogliomas 20 but this rate may be higher in the first years after AO diagnosis.

Conclusions
The present study suggested that STS poor survival largely results from more aggressive baseline characteristics and a more advanced disease at diagnosis. In these patients, reduced epileptogenicity and a higher proliferation index could lead to the diagnosis of large and symptomatic tumors in older patients. Future studies will have to determine how to optimally identify and treat AO patients at risk for poor outcome, yet surgical resection may be particularly important in this population.